Regulatory mutants affecting the levels of the asparagine biosynthetic enzyme, asparagine synthetase (AS), have been selected in cultured Chinese hamster ovary cells. All the mutants have elevated levels of AS, which are not regulatable by the endproduct of the pathway, asparagine. One type of mutant appears to have an alteration in the modification of certain tRNA's as well. Genetic analysis of the mutants has revealed they fall into at least 2 classes with respect to the behavior of AS in intraspecific hybrids. One mutant phenotype is dominant in hybrids and one is co-dominant. Experiments are now in progress to determine if these regulatory mutations are linked or unlinked to the structural gene for AS.